Clinicopathologic and experimental data suggest that astrocytes may be the most likely target for methotrexate (MTX) toxicity in the central nervous system (CNS). Both in vivo and in vitro experiments will be carried out to test this hypothesis. For in vivo studies, brain sections from Sprague-Dawley rats will be examined by light and electron microscopy at various times following intraperitoneal injection with varying doses of MTX. The glial fibrillary protein component will be studied by immunohistochemistry and special stains will be performed to compare MTX toxicity in astrocytes and other candidate targets in the CNS. The MTX effects will be evaluated in rats which have been fed with folic acid- deficient diet as well as following co- administration of the drug with folic acid, leucovorin and methyl tetrahydrofolate. MTX levels will be correlated with folate levels in brain following MTX treatment. For in vitro studies, primary astrocyte cultures derived from neonatal Fischer rat brains will be exposed to varying concentrations of MTX and examined by light and electron microscopy. Mitotic index will be determined. Growth curves will be constructed from daily viable cell counts using Trypan blue- exclusion method. To examine the mechanisms involved in MTX induced toxicity. MTX uptake will be measured and correlated with morphological changes, dihydrofolate reductase activity and folate levels in treated cultures. The effect of MTX on folate cofactor dependent metabolites (thymidylate, purine and amino acids), DNA protein synthesis, glutamate uptake, folyl polyglutamate synthetase activity and nucleotide levels will be investigated. The treated cultures will be studied in the presence or absence of glucose or sodium azide (an electron transport inhibitor) to determine the effect of cell energy metabolism on MTX activity. Cytokinetic studies using laser flow cytometry will be performed for cell cycle staging. The morphological, biochemical and functional effects of MTX will be evaluated in the presence or absence of several agents (folic acid, folinic acid, glycine, adenosine, thymidine, glutamate, glutamine and radiation) to see if the MTX effects can be modified. The data that will be obtained from this project will establish MTX toxicity in astrocytes, provide some insight on the pathogenetic mechanisms by which MTX causes CNS injury, and lead to future investigative efforts directed towards better therapeutic control and higher survival rate of cancer patients.